An assignment on THE IMMUNE RESPONSE IN SCHISTOSOMIASIS

Submitted with respect to: Dr. J. J. Hasnani Professor & Head Dept. of veterinary parasitology

Submitted by: Vijayata M.V.Sc (Scholar) Veterinary parasitology

Schistosoma japonicum
Definitive Hosts: Humans, wild mammals including dogs, cats, deer, primates, horses, swine, cattle. Least host specific. First Intermediate Host: aquatic snails. Second Intermediate Host: None Geographic Distribution: South East Asia Transmission to D.H.: Cercaria burrow into the skin. Location in D.H.: Veins of the small intestine

Schistosoma mansoni
Definitive Hosts: Humans, and many wild mammals including monkeys and rodents. First Intermediate Host: Aquatic snails Second Intermediate Host: None Geographic Distribution: Africa and South America Spread to South America with the slave trade.

Transmission to D.H.: Cercaria burrow into the skin. Location in D.H.: Portal veins of the large intestine.

Schistosoma haematobium
Definitive Hosts: Humans. Very host specific with no known reservoir hosts. First Intermediate Host: Aquatic snails Second Intermediate Host: None Geographic Distribution: northern Africa and small area of Middle East. Transmission to D.H.: Cercaria burrow into the skin. Location in the D.H.: veins of the urinary plexus (ureters, bladder, urethra).

Basic Considerations in Immunopathology

Most chronic morbidity in schistosomiasis is not due to the adult worms but is related to the Tcell-dependent immune response of the host, which is directed against schistosome eggs trapped in tissues, mainly in the liver and intestines in the case of the intestinal forms (S. japonicum and S. mansoni) and in the bladder in the case of S. haematobium. The trapped eggs secrete a range of molecules leading to a marked CD4_ T-cell programmed granulomatous inflammation involving eosinophils, monocytes, and lymphocytes, akin to a form of delayedtype hypersensitivity. Granulomas are also characterized by collagen deposition, and with the intestinal schistosomes, severe hepatic periportal (Symmers) fibrosis occurs. Much of the morbidity and mortality associated with this disease is attributable directly to the deposition of connective tissue elements in affected tissues. In mice, a predominantly T-helper 1 (Th1) reaction in the early stages of infection shifts to an egg-induced Th2-biased profile, and imbalances between these responses lead to severe lesions (114, 118, 138, 143, 161, 167).A notable accomplishment in the past few years was the identification of interleukin-13 (IL-13) and the IL-13 receptor complex as central regulators of disease progression in schistosomiasis (105, 125, 167). Similar regulatory control could be at the basis of fibrotic pathology in humans (1), although this has not yet been established.

Effector Mechanisms and Expression of Immunity in Animal Models of Schistosomiasis

A number of recent reviews have considered the immunobiology of schistosomiasis, including the nature of the host innate and adaptive responses to schistosomes and strategies used by the parasites to manipulate such responses (1, 26, 105,114, 118, 138, 167). Much of our understanding of the mammalian immune response to schistosomes is based on the use of genedisrupted (knockout) mice (51, 86, 125, 143, 167) and the immunization of mice, nonhuman primates, or other mammalian hosts with UV- or _-irradiated cercarial vaccines, with or without a subsequent challenge infection with nonattenuated cercariae (12, 41, 59, 75, 76, 132, 146). The attenuated larvae fail to mature into adult worms and do not produce eggs, so any results obtained are not confounded by egg-induced liver pathology. An even greater effect of triggering high-level resistanceagainst schistosome reinfection has been shown for mice treated with artemether, a methyl ether derivative of dihydroartemesinin, followed by challenge (11). This model may provide an alternative approach to irradiated vaccines for dissecting different immune responses as putative effector mechanisms during schistosome infection and protective responses against reinfection. In general, these studies have established that T-cell-mediated immunity is fundamental to acquired resistance to schistosomes in mice. Much of this protection was shown to be mediated by activated macrophages and, together with studies of cytokines, suggested that a vaccine that induced macrophage- activating Th1 cytokines (gamma interferon [IFN-_] and IL-2) may be beneficial in preventing schistosomiasis. However, repeated vaccination with irradiated cercariae produced incremental increases in Th2-mediated (IL-4 and IL-5 predominance) protection, which was transferable to nonvaccinated animals. Studies using B-cell-deficient and cytokine-deficient mice demonstrated that successful antischistosome vaccination required induction of strong Th1 and Th2 responses. Following infection by normal or radiation-attenuated cercariae, the predominant early immune response was Th1 mediated and aimed at the adult worm. Following egg deposition in tissues (at 6 weeks postinfection for S. mansoni and 4 to 5 weeks postinfection for S. japonicum), the Th1 response was diminished, being replaced by a prominent Th2-mediated phase. Indeed, it appears

that egg antigens are able to directly suppress the Th1 response (116, 118), a phenomenon which may also occur in humans. The Th2 response results in an increase in serum IL-5, massive bone and blood eosinophilia, and a granulomatous response aimed at the egg, resulting in collagen deposition, tissue fibrosis, and the disease manifestations of schistosomiasis. The precise role of eosinophils in the disease process in the mouse model of infection remains undetermined (141). The complexity of immune regulation and T-cell regulation in schistosome infection in mice is well recognized (98, 114, 143, 161), and this was further illustrated by a recent study by Walsh et al. (157), who highlighted a specific role for CTLA-4_ but not CD25_ cells in the regulation of Th2 responses in helminth infection. Furthermore, whereas the cytokine interplay during the development of protective immunity to the radiation-attenuated (RA) schistosome vaccine has been characterized extensively over recent years, the role of costimulatory molecules in the development of cell-mediated immunity is much less well understood. The importance of CD40/CD154 in vaccine-induced immunity was recently demonstrated (60), as it was shown that CD154_/_ mice exposed to RA schistosomes developed no protection to challenge infection, suggesting that protective immunity to the RA schistosome vaccine is CD154 dependent but is independent of (IL-12 orchestrated) cellular immune mechanisms in the lungs. As referred to earlier, in the case of S. japonicum, zoonotic mtransmission adds to the complexity of S. japonicum control programs but provides a unique opportunity to develop a transmissionblocking veterinary vaccine to help prevent human infection and disease. However, studies of protective immunity in bovine schistosome infections are few (101), and consequently, our knowledge of the immunology of schistosome infections in buffaloes and cattle is extremely limited. This is particularly the case for water buffaloes, for which immunological reagents for studying immune responses are scarce. Recent PZQ treatment and reinfection studies of bovines infected with S. japonicum in China have indicated that age-related resistance occurs in buffaloes but not cattle (159).Whether this self-cure phenomenon has an immunological basis has yet to be determined. Additional studies on the immunology of buffaloes and cattle represent an important area for future research and will be essential in selecting S. japonicum vaccine antigens and in defining the optimum route of immunization.

STRATEGIES FOR ANTISCHISTOSOME VACCINE DEVELOPMENT

Schistosomes do not replicate within their mammalian hosts. Consequently, a nonsterilizing naturally or vaccine-acquired immunity could significantly decrease human pathology and disease transmission. Vaccination against schistosomes can be targeted towards the prevention of infection and/or to the reduction of parasite fecundity. A reduction in worm numbers is the gold standard for antischistosome vaccine development, with the migrating schistosomulum stage likely to be the major vaccine target of protective immune responses (99, 163). However, as schistosome eggs are responsible for both pathology and transmission, a vaccine targeted at parasite fecundity and egg viability also appears entirely appropriate. While they regularly induce 50 to 70% (over 90% in some cases) protection in experimental animals and additional immunizations boost this level further, it may be premature to pursue RA schistosome vaccines for human use, but their development for veterinary application is feasible. The concept is proven, and many of the requisite techniques, although they require refining and upscaling, are published. Although technically challenging, there is a case for promoting the development of a

live, attenuated, cryopreserved schistosomulum vaccine for use against S. japonicum in buffaloes to reduce zoonotic transmission to humans in China (99). If successful, the veterinary vaccine could provide a paradigm for the development of antischistosome vaccines for human use. In addition, while the S. mansoni RA vaccine model has enabled the dissection of different immune responses as putative effector mechanisms (59) and raised hopes for the development of molecular vaccines, this has not equated to advances in the development of recombinant vaccines. Independent testing of six candidate S. mansoni antigens (glutathione S-transferase 28 [Sm28-GST], paramyosin, Ir-V5, triosephosphate isomerase, Sm23, and Sm14) in the mid1990s, orchestrated by a UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR/WHO) committee, resulted in protective responses being recorded, but the stated goal of consistent induction of 40% protection or better was not reached with any of the antigens tested, highlighting the possible negative influence of insufficient antigen stability and the need for standardized and effective adjuvant formulations (9). Furthermore, of these six antigens, only one (Sm23) is exposed on the apical membrane surface of the parasite (165), although it is not one of the more abundant apical membrane proteins on the parasite surface (17). Also, the failure to develop an efficacious schistosome vaccine can be attributed in part to the complex immunoevasive strategies used by schistosomes to avoid elimination from their intravascular environment (118). Nevertheless, convincing arguments still support the likelihood that effective vaccines against the various schistosome species can be developed (9); first, as discussed above, irradiated cercariae regularly induce high levels of protection in experimental animals, and additional immunizations boost this level further; second, as we have emphasized, endemic human populations develop various degrees of resistance, both naturally and drug-induced; and third, veterinary antihelminth recombinant vaccines against cestode platyhelminths have been developed successfully and applied in practice (35). The optimism sparked by these arguments has resulted in the discovery of a large number of schistosome antigens (utilizing the almost-complete genome sequence), and additional candidates are now being found through proteomic approaches (16, 17); these two dynamic areas of schistosome molecular biology are explored further below. However, antigen identification and successful protective results are of little value if recombinant proteins cannot be produced easily (and cheaply) with good manufacturing practice (GMP). Even the best protective results are no guarantee for ultimate success, and the scaling up of antigen production can be every bit as challenging as any immunological investigation. This was underscored when several of the nfrontline candidates chosen by the TDR/WHO committee discussed above had to be abandoned because, in addition to the low independent testing efficacy recorded, hurdles in consistent protein production could not be overcome. Nevertheless, as discussed below, there is still considerable interest in developing these and other molecules as antischistosome vaccines. Compromises may be necessary, however, because as emphasized by Bergquist and colleagues, we might eventually have to settle for moderately effective antigens, but where the scaled-up GMP versions do not pose a problem. Despite the extra costs of scaling-up production of all antigens under consideration, it is a selection criterion in assessing vaccine candidacy that cannot be avoided (9). A number of recent studies, particularly on S. japonicum,have utilized plasmid DNA vaccines to deliver protective antigens. DNA vaccines generate both T-cell and B-cell (or antibody-mediated) immune responses and are thus particularly appealing for schistosome vaccine development. The preparation and production of DNA vaccines are convenient and costeffective, and they can even be used in the fieldwithout a cold chain. Another advantage of applying DNA vaccines compared to other approaches is the possibility oftargeting the in vivo

expressed recombinant antigen to different cell compartments. Furthermore, methods such as primeboost regimens and the use of adjuvants (such as IL-12) in combination with a DNA vaccine can enhance its protective effectiveness. The advantages and disadvantages of plasmid DNA vaccination, the strategies employed for DNA vaccine delivery, and technological and clinical advances in the area have been reviewed recently (18, 148).

CURRENT STATUS OF VACCINE DEVELOPMENT FOR S. MANSONI AND S. HAEMATOBIUM

Major Candidate Vaccines and Their Protective Efficacies

Tetraspanins.
Tetraspanins are four-transmembrane-domainm proteins found on the surfaces of eukaryotic cells, including B and T cells. They have two extracellular loops, including a short loop 1 of 17 to 22 residues (EC-1) with little tertiary structure and a larger, 70- to 90-residue loop 2 (EC-2),

which as four or six cysteines that form two or three disulfide bonds (Fig. 2). In general, the extracellular loops mediate specific protein-protein interactions with laterally associated proteins or, in some cases, known ligands (reviewed in reference 90).

Sm28/Sh28 GST:Sm28-GST has GST properties and is expressed in subtegumental tissues of most developmental stages of the parasite (120). Vaccination of semipermissive rats and permissive hamsters with recombinant Sm28-GST resulted in significant reductions of worms (7), kickstarting a 20-year program on Sm28- and Sh28-GSTs as vaccine antigens. Primate trials were conducted and showed an antifecundity effect (15), and an anti-Sm28 monoclonal antibody showed antifecundity and anti-egg embryonation effects (168). This led to the clinical testing of Sh28-GST in people and the description of its immunogenicity and induction of antibodies capable of neutralizing the enzymatic activity of the recombinant protein (26, 27). Unfortunately, there are no data available on the efficacy of this vaccine in phase II clinical trials.

Smp80 calpain:Calpain is a calcium-activated neutral cysteine protease. The calpain large subunit was first discovered from S. mansoni by immunoscreening of a lambda phage cDNA library with sera from infected humans (5). Calpain was immunolocalized to the tegument and underlying musculature of adult worms and was shown to be involved in surface membrane turnover (135) and to be associated with the inner tegument membrane (17). Calpain was shown to be the target of a protective CD4_ T-cell clone that induced peritoneal macrophages from syngeneic recipients to kill schistosomula inmvitro (69). In addition, mouse recipients of this T-cell clone displayed significant resistance against cercarial challenge, making calpain the first vaccine antigen identified on the basis of T-cell reactivity. The large subunit of calpain, called Sm-p80, was expressed in baculovirus, and the semipurified protein induced 29 to 39% reductions in worm burdens (65). Subsequent efforts to improve the efficacy of this vaccine have focused on DNA vaccine constructs, with and without Th1-type cytokine cDNAs, in mice and baboons (134).

SOD:Granulocytes release oxygen radicals that are toxic for S. mansoni, and exogenous superoxide dismutase (SOD) inhibited granulocyte toxicity for egg metabolic activity and hatching (77). A cDNA encoding a SOD with a signal peptide was cloned from S. mansoni, and its protein product was recognized by sera from infected humans (136). A cDNA encoding a cytosolic SOD (CT-SOD) was then identified (63), and both SODs were immunolocalized to the tegument and subtegumental tissues (64, 104). Proteomic studies have since shown that SOD is localized below the tegument plasma membrane (16, 150). Vaccination experiments using the recombinant SOD proteins have not been reported, but CT-SOD and a partial sequence encoding the structural protein filamin showed promise as DNA vaccines, resulting in significant reductions in adult S. mansoni in a murine challenge model(133).

Paramyosin:Paramyosin is a 97-kDa myofibrillar protein with a coiled-coil structure and is found exclusively in invertebrates. It is expressed on the surface tegument of lung-stage schistosomula in the

penetration glands of cercariae (reviewed in reference 55) and may function as a receptor for Fc (94). The vaccine efficacy of paramyosin against S. mansoni was first described in the 1980s; mice immunized intradermally with S. mansoni extracts and Mycobacterium bovis BCG adjuvant were significantly protected against subsequent infection, and antibodies predominantly recognized paramyosin (85). Vaccination of mice with native and recombinant paramyosin was then shown to provide modest (26 to 33%) but significant protection against challenge infection with S. mansoni (117).

FABPs:The S. mansoni fatty acid binding protein (FABP), Sm14, is a cytosolic protein expressed in the basal lamella of the tegument and the gut epithelium (21). Sm14 has been assessed thoroughly as a recombinant protein vaccine and, to a lesser extent, as a DNA vaccine. Despite a high efficacy of recombinant Sm14 protein in mouse vaccine trials (144), Sm14 failed to induce protection levels of _40% when tested in other laboratories (49) and as part of the WHO/TDR-sponsored trials (8). Coadministration of recombinant Sm14 protein with either IL-12 (49) or tetanus toxin fragment C (2) boosted protection. Immunization of mice with recombinant Sm14 expressed in Mycobacterium bovis BCG showed no induction of specific antibodies to Sm14, but splenocytes from vaccinated mice produced IFN-_ upon stimulation with recombinant Sm14. Moreover, mice that were vaccinated once with Sm14-BCG and then challenged with S. mansoni cercariae showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of recombinant Sm14 protein (151).

CURRENT STATUS OF VACCINE DEVELOPMENT FOR S. JAPONICUM Major Candidate Vaccines and Their Protective Efficacies

Considerable efforts have been aimed at the identification of relevant S. japonicum antigens that may be involved in inducing protective immune responses, with a view to developing them further as viable vaccines. Vaccination can be targeted either towards the prevention of schistosome infection or to the reduction of parasite fecundity. A reduction in worm numbers is the gold standard for antischistosome vaccine development, but because schistosome eggs are responsible for both pathology and transmission, a vaccine targeted at parasite fecundity and egg viability is also relevant. Some of the leading S. japonicum vaccine candidates (as recombinant protein and/or DNA vaccines) are discussed below; the protective efficacies of these and other molecules in different host animals are summarized in Tables 2 and 3. The majority are membrane proteins, muscle components, or enzymes, and further details of the characteristics and efficacies of these and other vaccine candidates can be found elsewhere (99, 101, 166). Sj26GST. The GSTs are a group of enzyme isoforms that catalyze the detoxification of lipophilic molecules by thio-conjugation. In light of their physiological importance, a number of research groups have investigated the potential of the GSTs as vaccine targets for S. mansoni and S. haematobium (see above). Some encouraging data are available for the protective efficacy of Sj28GST against S. japonicum in different mammalian hosts in China (99, 166) (Tables 2 and 3), but more recent work has focused on the 26-kDa isoform. Recombinant Sj26GST (rSj26GST) induces a pronounced antifecundity effect as well as a moderate but significant level of

protection in terms of reduced worm burdens in mice, sheep, cattle, and pigs following challenge infection with S. japonicum (166). Similar levels of vaccine efficacy were obtained in water buffaloes vaccinated with purified rSj26GST (166). Anti-Sj26GST antibodies were produced in the immunized water buffaloes, and following challenge with S. japonicum cercariae, the typical antifecundity effect was manifest, characterized by significant decreases in fecal egg output and in eggs deposited in host tissues, with those in the liver and intestine being reduced about 50%. In addition to the antifecundity effect, rSjc26GST reduced the egg-hatching capacity of S. japonicum eggs into viable miracidia by nearly 40%. Recent field trials have demonstrated that the protective effect of the rSj26GST vaccine against S. japonicum can be maintained in cattle and water buffaloes for at least 12 months (99, 166).

Paramyosin (Sj97).
Paramyosin is a 97-kDa myofibrillar protein with a coiled-coil structure and is found exclusively in invertebrates. It is expressed on adults and the tegumental surfaces of lung-stage schistosomula and appears to be multifunctional. It may act as a receptor for Fc (94), and an exogenous form inhibits activation of the terminal pathway of complement, implying an important immunomodulatory role in schistosomiasis (55). Native and recombinant paramyosin (Sj97) proteins confer protection against S. japonicum in mice,water buffaloes, and other mammalian hosts (166). Furthermore, recent studies of human antibody isotype (109) and Th2 cytokine responses to Sj97 add further support to this molecule as a leading vaccine candidate against S. japonicum (89). Unfortunately, a major challenge with Sj97 is its poor expression in soluble form, probably due to its coiled-coil structure and its large size. To improve its expression and to identify protective epitopes on paramyosin, the published Sj97 ( sequence was recently redesigned using Pichia codon usage and divided into four overlapping fragments (fragments 1, 2, 3, and 4), of 747, 651, 669, and 678 bp, respectively (172). These gene fragments were synthesized and expressed in Pichia pastoris (fragments 2 and 3) or Escherichia coli (fragments 1 and 4). The recombinant proteins were produced at high levels and purified, and BALB/c mice were immunized with the purified proteins formulated in the adjuvant Quil A. The protein fragments were highly immunogenic, inducing high, though variable, enzyme-linked immunosorbent assay antibody titers, and each was shown to resemble native paramyosin in terms of its recognition by the antifragment antibodies in Western blots. Promising protective efficacy in terms of significant reductions in worm burdens, worm pair numbers, and liver eggs in vaccinated mice resulted, but there was no apparent correlation between the antibody titers generated and protective efficacy, as all fragments produced effective but similar levels of protection.These fragments now need to be tested further for protective potency, both separately and in combination, in larger animals, including water buffaloes. Full-length DNA vaccines coding for Sj97 have been shown to induce protective immunity in mice (30), confirming previous studies (99).

SVLBP.
An expressed sequence tag of S. japonicum encoding an S. japonicum very-low-density lipoprotein binding protein (SVLBP; molecular size, 20 kDa) was reported to be membrane associated and located in the teguments and subteguments of adult male schistosomes (45). Given that SVLBP may play an essential role in lipid acquisition by the parasite and/or in signal transduction pathways, its further investigation for development as a novel antischistosomal intervention waswarranted. Accordingly, Gan et al. (53) used affinity-purified recombinant SVLBP (rSVLBP) to vaccinate mice. The worm numbers and egg numbers recovered from the veins and livers of the immunized mice were 33.5% and 47.6% lower, respectively,than those from control mice. There was also a marked increase in the antibody response in vaccinated mice: the titers of IgG1, IgG2a, and IgG2b of the vaccinated group were significantly higher than those of the controls. In a comparison of the reactivities of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of individuals. Notably, the average titer of anti-rSVLBP antibody in sera from fecal egg-negative individuals was significantly higher than that in sera from fecal eggpositive individuals, which may reflect SVLBP-specific protection. These results suggest that the parasite tegumental protein SVLBP is a promising candi- date for further investigation as a

vaccine antigen for use against schistosomiasis japonica, but further testing is now required to assess its true value.

Serine protease inhibitor (serpin).

Serine proteinase inhibitors(serpins) represent an important superfamily of endogenous inhibitors that regulate proteolytic events active in a variety of physiological functions. Yan et al. (169) used immunological screening of an S. japonicum adult worm cDNA expression library with sera of Microtus fortis, a naturally resistant rodent host, and identified one clone that encoded a homologous to those of the serpin superfamily. The full-length sequence encoding S. japonicum serpin was amplified from adult worm cDNA by using 5_ rapid amplification of cDNA endsPCR (5_ RACE-PCR) and subsequently cloned into the prokaryotic expression vector pET28c. The full-length S. japonicum serpin fusion protein with a His tag was expressed in E. coli, purified by affinity chromatography, and used to immunize rabbits. The S. japonicum serpin is located on the tegument in S. japonicum adult worms. C57BL/6 mice immunized with S. japonicum serpin induced the production of high levels of specific IgE and IgG1 subclass antibodies as well as a marked IL-4 response. Lymphocyte surface marker analysis revealed the proliferation of CD19-expressing B cells, indicating a predominant Th2-type response to S. japonicum serpin. Immunized mice developed moderate protection against infection with S. japonicum, as demonstrated by 36 and 39% reductions nin the recovery of adult worms and eggs, respectively.Further vaccine-challenge experiments with S. japonicum serpin, modifying the delivery of the vaccine and testing differentbadjuvant formulations, will enable a better assessment of its potential as a vaccine candidate.

SjTPI.
The glycolytic pathway enzyme triose-phosphate isomerase (TPI) is found in each cell of each stage of the schistosome life cycle, and the S. mansoni enzyme (SmTPI) has long been targeted as a schistosomiasis vaccine candidate. Since the two schistosome TPI sequences are very similar (84% identity), it was logical to assess the protective efficacy of S. japonicum TPI (SjTPI). Encouraging results were obtained with Chinese SjTPI (SjCTPI) plasmid DNA in early experiments on mice (177), but to examine the transmission-blockingnpotential in larger animals, Zhu et al. (178) determined its vaccine efficacy in naive pigs. Pigs were vaccinated with the TPI DNA plasmid, alone or in conjunction with IL-12 plasmids, via intramuscular injection. Pigs vaccinated with SjCTPI DNA alone had adult worm burdens that were reduced 48.3%; a further decrease in adult worm burdens was not seen in then group vaccinated with SjCTPI DNA in conjunction with IL-12 (46.2% reduction). The SjCTPI DNA vaccines had a more pronounced effect on reducing female worm burdens, i.e., 53.6% for SjCTPI alone and 59.6% for SjCTPI plus IL-12. Vaccination with SjCTPI DNA reduced liver egg numbers 49.4%, and this response was significantly enhanced by the addition of IL-12 (65.8% reduction in liver eggs). In addition to the dramatic protective effects seen in vaccinated pigs, it was also noted that granuloma size was reduced 42% in both groups. Coimmunization with a DNA plasmid of SjCTPI fused to heat shock protein 70 (SjCTPI-Hsp70) and IL-12 DNA induced protective immunity against experimental S. japonicum infection in water buffaloes (170). Although these data are encouraging, further extensive experimental and field-based natural challenge trials on bovines, particularly water buffaloes, in China are now required to determine whether vaccination with the SjCTPI DNA vaccine will likely reduce transmission by reducing adult worm burdens and worm egg output, with simultaneous reduction of hepatic egg-associated pathology.

Twenty-three-kilodalton integral membrane protein (Sj23).

As with TPI, the tetraspanin integral membrane protein (Sm/Sj23) was identified as a major vaccine candidate some years ago, first against schistosomiasis mansoni and then against schistosomiasis japonica. The Chinese S. japonicum form (SjC23) was initially shown to induce protection in mice as a synthetic peptide vaccine and then, as a plasmid DNA vaccine, also induced protection in mice, sheep, pigs, and water buffaloes. Overall, the results from extensive plasmid DNA vaccine studies indicated that vaccination with SjC23 DNA not only induced significant reductions in worm and egg burdens but also significantly reduced the size of egg granulomas; thus, like SjCTPI, SjC23 produced an antipathology effect as well. The protective effect of the SjC23 plasmid DNA vaccine was enhanced with IL-12 in pigs (175) and mice (53, 176) and by a CpG immunostimulatory sequence in mice (173). As with the other candidate vaccines, extensive large animal field trials are now required to determine the precise protective potency of SjC23, with or without IL-12 or CpG.

SjFABP (Sj14).
Like other parasitic helminths, schistosomes are unable to synthesize long-chain fatty acids or sterols and hence are completely dependent on the host for these components. FABPs are critical for schistosomes to take up fatty acids from host blood as essential nutrients and are thus prime targets for both vaccination and drug development. The 14- kDa FABP of Chinese S. japonicum (SjFABPc) has at least eight different variants encoded by a single-copy polymorphic gene, and it is particularly important to S. japonicum for uptake, transport, and compartmentalization of host-derived fatty acids, playing a vital role in the physiology and survival of the parasite. Several Chinese groups have obtained encouraging protection in mice by using SjFABPc, both as a recombinant protein and as a plasmid DNA vaccine. Especially noteworthy are the studies by Liu et al. (92), who expressed SjFABPc in E. coli and in baculovirus/silkworm systems. The recombinant protein from E. coli was a 41-kDa GST fusion protein (rSj14/GST), which could be purified by glutathioneagarose affinity chromatography, with a yield of 25 mg/liter E. coli culture. The recombinant protein from the baculovirus/ silkworm system was an 18-kDa fusion protein (rSj14/His), which could be purified by Ni-nitrilotriacetic acid resin chromatography, with a yield of 3.5 mg per silkworm larva. Both rSj14/GST and rSj14/His were recognized by S. japonicuminfected mouse sera and anti-rSj14/GST mouse sera in Western blots. The purified recombinant protein was immunogenic in several mammalian host species, and 34.3%, 31.9%, and 59.2% worm reductions were obtained in Sj14/GST-vaccinated Kunming mice, Wistar rats, and sheep, respectively, compared to nonvaccinated control groups. Worm reductions of 48.8% and 49.0% were recorded for BALB/c mice immunized with Sj14/His compared to nonvaccinated and BCG-vaccinated groups, respectively. Taken together, these results emphasize the promise of SjFABPc as a candidate vaccine for schistosomiasis japonica, particularly as no adjuvant was used in the rat and sheep vaccination experiments. Another group (174) studied the protective efficacy of SjFABPc as a DNA vaccine enhanced by IL-12 in mice challenged with S. japonicum. They showed that IL-12 drives the immune response toward a Th1 direction and enhances the protective effect of the vaccine. Bivalent DNA vaccine constructs encoding SjFABPc and Sj23 provided higher levels of protective efficacy against S. japonicum in mice than those obtained with the univalent DNA vaccines (171).

Calpain.

Calpain is efficacious against S. mansoni and is also recognized as an encouraging vaccine candidate against schistosomiasis japonica (110). When BALB/c mice were immunized with purified recombinant S. japonicum calpain emulsified in complete Freunds adjuvant, significant reductions in the number of recovered worms (Table 2) and also in egg production per female worm were observed. Furthermore, raised levels of inducible nitric oxide synthase expression were observed in immunized mice, while adhesion of peritoneal exudate cells also occurred in the presence of sera from immunized mice, suggesting the involvement of both cellular and humoral protective mechanisms. In addition, spleen cells from the immunized mice showed enhanced production of IFN-_ by activated CD4_ T cells, and subsequent work with calpainspecific mouse T-cell hybridomas identified the T-cell epitope (EQLKIYAQRC) involved (112). Localization studies have shown that calpain is present in the penetration glands and in the secretions of cercariae (83).

NEW ANTIGEN DISCOVERY FOR VACCINES AGAINST SCHISTOSOMES

The current Schistosoma vaccine candidates may prove not to be the most effective. It is important to identify new target antigens and to explore alternative vaccination strategies to improve vaccine efficacy. The available schistosome antigens and prototype vaccine formulations induce 40 to 50% protection in animals, at best, using the standard readouts of reduced worm burden or egg production and viability. This apparent efficacy ceiling (for antigen combinations as well) has proved a significant roadblock to success. Accordingly, the current model vaccines may not be sufficiently protective or characterized by reproducible efficacy. Difficulties in obtaining good expression levels and in scaling up production according to good laboratory practice/GMP standards for the limited number of antigens selected have turned out to

be another major obstacle. Some frontline candidates have suffered from difficulties in scale-up production according to good laboratory practice/GMP standards and have been dropped. The feasibility of large-scale production should be a prime selection criterion in assessing the vaccine candidacy of schistosome antigens (9). Mining and functional annotation of the greatly expanded S. mansoni (154) and S. japonicum (67) transcriptomes and their npublic accessibility through public databases, in combination with postgenomic technologies, including DNA microarray profiling, proteomics, glycomics, and immunomics, have the potential to identify a new generation of potential vaccine target molecules that may induce greater potency than the current candidate schistosome antigens. Perhaps the most important advance in postgenomics forschistosomiasis has been the successful application of RNA interference (RNAi) to schistosomes (20, 82). These studies have had (and will continue to have) an enormous impact on our ability to determine the functions of schistosome genes/proteins and which ones are essential for survival and reproduction. Silencing the expression of numerous S. mansoni genes has resulted in phenotypic changes (34, 52, 84), highlighting their importance as targets for vaccines and new drugs. Genome-wide RNAi has been used to assess the functions of most genes from the free-living nematode Caenorhabditis elegans (reviewed in reference 122), and the eventual application of this technology to schistosomes will revolutionize the way we search for (and test) vaccine and drugtargets. Molecules containing signal peptides and signal anchors as predictors of excretory-secretory products, including enzymes, and components exposed on the schistosome epithelial surfaces (including receptors) that interact directly with the host immune system are highly relevant targets for schistosome vaccines (28, 71, 95, 137). The burgeoning area of schistosome genomics and postgenomic research has been reviewed extensively (62, 66, 102, 103, 123, 162, 164), but one important point that needs to be made is that the majority of studies have been undertaken on S. mansoni and S. japonicum; there is almost a complete absence of transcriptome/genome information for S. haematobium, and this is clearly an important area for futurestudy. There is an abundance of reports on schistosome antigens (from different anatomic locations within different stages of the parasite) that provide in the vicinity of 30% reductions in adult worm burdens. The tegument is where many researchers have focused their efforts, but it is those few tegument proteins which are truly exposed to the host immune system in a live wormthe tegument plasma membrane proteinswhich, in our opinion, should be a major focus for future vaccinology efforts (95). Where investigated, membrane-spanning proteins of the tegument, e.g., the tetraspanins and Sm29, have shown great promise (Table 1). This subset of exposed proteins (16,17), which present extracellular regions of various sizes outside the cell, should attract much more attention in the future, and we advocate that efforts of schistosomiasis vaccine laboratories would be better invested in developing methods to produce and deliver schistosome surface antigens (see below) or secreted molecules than in continuing to identify new intracellular antigens that show modest protection at best.

ANTIGEN FORMULATION AND DELIVERY OF VACCINES AGAINST SCHISTOSOMES

Extracellular vaccine candidates need to be expressed in bacteria or eukaryotic expression systems. Many of the selected targets are likely to require processing through the endoplasmic reticulum by virtue of their expression sites in theparasite (i.e., secreted or anchored in the tegument), and this may prove challenging. An additional important consideration is that antigen

identification and successful protective results are of little value if GMP cannot be applied for scaling up of production of any vaccine candidate (9). The selection of a suitable adjuvant and delivery system to aid in the stimulation of the appropriate immune response is a critical step in the path to the development and employment of successful antischistosome vaccines, and a number of approaches have been tested, with some success. Traditional approaches have seen Freunds adjuvants used when antigens are first being assessed as vaccines in the mouse model. It must be remembered, however, that Freunds complete adjuvant, although the mainstay of immunological adjuvants in research for decades, is not suitable for human application, as it can produce a number of undesirable side effects that include the formation of local inflammatory lesions at the site of the injection that can result in chronic granulomas and abscesses. Once efficacy has been proven with Freunds adjuvants, other adjuvants, particularly those that are licensed (or have the potential for licensing) for human use, should be used to formulate an antigen. Less conventional or less widely used approaches have been explored as adjuvants for schistosome vaccines, including live Salmonella (113), tetanus toxin (2), filamentous phages (124), recombinant Mycobacterium bovis BCG (151, 152), nanoparticles (46), and various methods of mucosal delivery (88, 121, 140). Before a well-informed decision can be made on adjuvant selection, a comprehensive understanding of the desired immune response (phenotype) is necessary. This, in turn, implies that the immune parameters required to obtain optimal protection are known. For human schistosomiasis, this is not the case. For example, very few people develop natural resistance to the parasite in the absence of repeated antihelminthic therapy.We advocate the use of such cohorts to guide vaccine development (both antigen discovery and the phenotype of the protective response), but in reality, a schistosome vaccine will be delivered as part of an integrated control package that involves PZQ treatment vaccination. Therefore, should we look more to the people who develop resistance to reinfection after PZQ therapy(158)? These two groups of individuals have very different immune responses to different antigens on different stages of the parasites (32, 108, 158). All of this information is relevant, complicated, to deciding how best to formulate and deliver a vaccine for human schistosomiasis. If we are to target Tolllike receptors (TLRs) on antigen-presenting cells that induce a Th1 response, such as TLR-9, then adjuvants such as unmethylated CpG dinucleotides are attractive, and although not yet widely used for schistosomiasis vaccinology, these adjuvants are showing promise for experimental vaccines against other parasites (38). Indeed, the PR individuals identified in Brazil (32), who were utilized to identify two new tegument antigens (28, 147), mount a vigorous Th1 response to schistosomulum surface antigens, making CpGs a potentially attractive adjuvant for these vaccines. CpGs are being used in conjunction with more conventional adjuvants, such as alum, which induces a more Th2-like immune response. For the diphtheria-tetanus-pertussis vaccine, which is currently formulated with alum, the addition of CpGs reduced the total IgE levels and increased anti-pertussis toxin IgG2a in comparison with the ordinary diphtheriatetanus-pertussis-alum vaccine(139). If a mixed Th1/Th2 response is optimal for a schistosomiasis vaccine, combination adjuvants such as alum-CpG seem to be a suitable way forward.

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